Rapid proteolytic degradation is a major mechanism for modulating the intracellular concentrations of regulatory proteins and affects developmental pathways, stress responses, metabolic adaptation, and essential cell cycle control processes. In vivo, most protein degradation is energy-dependent. Our research is focused on the ATP-dependent Clp and Lon proteases of E. coli and their homologs in mitochondria of human cells. The gene for a human Lon protease has been mapped to chromosome 19, region 13.3, and a cosmid clone of the genomic DNA has been isolated. A human cDNA encoding a homolog of ClpP has been isolated. Human ClpP is more than 50 percent identical to the bacterial protease at the amino acid level. Human ClpP expressed in E. coli and purified appears to have an oligomeric structure similar to the double-donut formed by E. coli ClpP but is not activated by E. coli ClpA. Electron microscopic studies show E. coli ClpAP has a structure analogous to that of the eukaryotic 26S proteasome. ClpP, the proteolytic core of the enzyme, is composed of superimposed seven-membered rings, whereas ClpA, the ATP-dependent component with protein unfolding activity, is a bi-lobed ring of only six subunits. In the ClpAP complex, a hexamer of ClpA is bound to each heptameric face of ClpP. Changes in these asymmetric interactions between subunits during the catalytic cycle may be important for unfolding or translocation of substrates during ATP-dependent proteolysis. A ClpA mutant with a lysine to glutamine substitution in the ATP-binding site of the first domain forms mixed hexamers with wild-type ClpA. Exchange studies with the mutant ClpA indicate that the half-time for subunit dissociation of ClpA under assay conditions is more than 8 min. Since the turnover number for peptide bond cleavage during proteolysis is at least 15 per min, multiple rounds of degradation occur without dissociation of ClpAP. Purified E. coli Lon protease requires ATP hydrolysis to degrade purified native CcdA, a physiological substrate for Lon, whereas degradation of a truncated form of CcdA by Lon is activated by nucleotide binding alone. The same peptide bonds are cleaved in each protein. Thus, ATP hydrolysis does not affect the specificity of the interaction between substrates and the proteolytic active site, but is required for unfolding or conformational alteration of the protein. Interaction with CcdB completely protects CcdA from degradation by Lon, demonstrating the importance of macromolecular interactions in determining the stability of regulatory proteins in vivo.